Method for preparing recombinant heterocarpine

ABSTRACT

A process for preparing recombinant heterocarpine with the complete sequence of heterocarpine (SEQ. ID. NO. 10), expression vectors comprising a polynucleotide encoding for heterocarpine, host cells transformed or transfected by said expression vectors as well as a process for obtaining heterocarpine by means of said transformed or transfected host cells are described where in the recombinant heterocarpine obtained is used to treat cancer.

A subject of the invention is a process for preparing recombinantheterocarpine.

Heterocarpine is a protein with anti-cancer properties described for thefirst time by the Applicant in Patent Application PCT WO 02/068461. Thisisolated protein has a molecular mass of approximately 90.9 kDa,comprises fragments of peptide sequences SEQ. ID. NO. 1, SEQ. ID. NO. 2and SEQ. ID. NO. 3 (see the part of the description reserved for thesequence listing) and is capable of being obtained by extraction fromPilocarpus heterophyllus plant cells cultured in vitro as described inthe abovementioned patent application. However, the complete sequence ofthis protein remained unknown up to now, to the extent that cloning hadnot been carried out.

The present application now describes polynucleotides which can serve asa primer for cloning heterocarpine, the DNA encoding for heterocarpine,the mRNA corresponding to heterocarpine, expression vectors containingsaid mRNA, host cells transformed or transfected with these vectors aswell as a process for preparing recombinant heterocarpine.

A subject of the present invention is therefore firstly an isolatedpolynucleotide comprising the polynucleotide sequence SEQ. ID. NO. 8.Preferably, said isolated polynucleotide consists of the polynucleotidesequence SEQ. ID. NO. 8.

A subject of the present invention is also an anti-sense polynucleotidecomprising the sequence complementary to that of said isolatedpolynucleotide comprising the polynucleotide sequence SEQ. ID. NO. 8.Preferably, said anti-sense polynucleotide consists of the sequencecomplementary to the polynucleotide sequence SEQ. ID. NO. 8.

The invention also relates to an isolated polynucleotide comprising thepolynucleotide sequence SEQ. ID. NO. 8 or one of the fragments of thelatter, said polynucleotide being such that it encodes a polypeptidehaving at least one immunological and/or biological activitycharacteristic of a protein binding human GHRH and being associated withthe modulation of cell proliferation. Preferably, said isolatedpolynucleotide is such that it consists of the polynucleotide sequenceSEQ. ID. NO. 8 or one of the fragments of the latter.

In particular, the invention therefore relates to the isolatedpolynucleotide of nucleotide sequence SEQ. ID. NO. 9 or the isolatedpolynucleotide of nucleotide sequence complementary to the nucleotidesequence SEQ. ID. NO. 9.

Heterocarpine, in other words the protein of sequence SEQ. ID. NO. 10,is encoded by the fragment of the polynucleotide of polynucleotidesequence SEQ. ID. NO. 8 contained between the bases at positions 115(initiation codon ATG encoding for a methionine) and 2437 (stop codonUAA), i.e. by the polynucleotide sequence SEQ. ID. NO. 9.

The invention therefore also relates to an expression vector comprisingan isolated polynucleotide comprising the polynucleotide sequence SEQ.ID. NO. 8 or one of the fragments of the latter or the sequencecomplementary to the polynucleotide sequence SEQ. ID. NO. 8 or one ofthe fragments of the latter, the polypeptide encoded by said isolatedpolynucleotide having at least one immunological and/or biologicalactivity characteristic of a protein binding human GHRH and beingassociated with the modulation of cell proliferation. Preferably, saidexpression vector will comprise the polynucleotide sequence SEQ. ID. NO.9 or the sequence complementary to the polynucleotide sequence SEQ. ID.NO. 9.

The invention similarly relates to a host cell transformed ortransfected with said expression vector.

The invention also relates to an isolated polypeptide comprising apolypeptide of sequence SEQ. ID. NO. 14 or one of its fragments, saidisolated polypeptide having at least one immunological and/or biologicalactivity characteristic of a protein binding human GHRH and beingassociated with the modulation of cell proliferation. Preferably, saidisolated polypeptide will consist of a polypeptide of sequence SEQ. ID.NO. 14 or one of its fragments. In particular, said polypeptide willhave the sequence SEQ. ID. NO. 14.

A subject of the invention is also a monoclonal antibody, or anantigen-binding fragment of the latter, which specifically binds anisolated polypeptide as described previously, and in particular amonoclonal antibody, or an antigen-binding fragment of the latter, whichspecifically binds the protein of sequence SEQ. ID. NO. 14 but not theprotein of sequence SEQ. ID. NO. 10.

The invention also relates to, as a medicament, an isolatedpolynucleotide comprising:

-   -   the polynucleotide sequence SEQ. ID. NO. 8 or one of its        fragments, or    -   the polynucleotide sequence SEQ. ID. NO. 9 or one of its        fragments,

said isolated polynucleotide being such that it encodes an isolatedpolypeptide having at least one immunological and/or biological activitycharacteristic of a protein binding human GHRH and being associated withthe modulation of cell proliferation.

Preferably, the isolated polynucleotide will consist of thepolynucleotide of polynucleotide sequence SEQ. ID. NO. 8 or one of itsfragments, or the polynucleotide of polynucleotide sequence SEQ. ID. NO.9 or one of its fragments.

In particular, said isolated polynucleotide will consist of thepolynucleotide of polynucleotide sequence SEQ. ID. NO. 8 or thepolynucleotide of polynucleotide sequence SEQ. ID. NO. 9.

Moreover, said isolated polynucleotide used as a medicament ispreferably present in a viral vector, said viral vector being forexample selected from the group consisting of an adenovirus, anassociated adenovirus, a retrovirus and a pox virus.

The invention also relates to, as a medicament, an isolated polypeptidecomprising a polypeptide of sequence SEQ. ID. NO. 14 or one of itsfragments, said isolated polypeptide having at least one immunologicaland/or biological activity characteristic of a protein binding humanGHRH and being associated with the modulation of cell proliferation.Preferably, said isolated polypeptide will consist of a polypeptide ofsequence SEQ. ID. NO. 14 or one of its fragments. In particular, saidpolypeptide will have the sequence SEQ. ID. NO. 14.

The invention also relates to, as a medicament, a monoclonal antibody,or an antigen-binding fragment of the latter, which specifically bindsan isolated polypeptide comprising the protein encoded by thepolynucleotide sequence SEQ. ID. NO. 9 or one of the fragments of thelatter or by a sequence complementary to the polynucleotide sequenceSEQ. ID. NO. 9 or one of the fragments of the latter, said isolatedpolypeptide having at least one immunological and/or biological activitycharacteristic of a protein binding human GHRH and being associated withthe modulation of cell proliferation. Preferably, said monoclonalantibody or said fragment of the antigen of the latter specificallybinds an isolated polypeptide consisting of the protein encoded by thepolynucleotide sequence SEQ. ID. NO. 9 or one of the fragments of thelatter or by a sequence complementary to the polynucleotide sequenceSEQ. ID. NO. 9 or one of the fragments of the latter. Yet morepreferentially, said monoclonal antibody or said fragment of the antigenof the latter specifically binds an isolated polypeptide consisting ofthe protein encoded by the polynucleotide sequence SEQ. ID. NO. 9 or bya sequence complementary to the polynucleotide sequence SEQ. ID. NO.9(in other words the protein of sequence SEQ. ID. NO. 10).

Another subject of the invention is a pharmaceutical compositioncomprising, as active ingredient, an isolated polynucleotide comprising:

-   -   the polynucleotide sequence SEQ. ID. NO. 8 or one of its        fragments, or    -   the polynucleotide sequence SEQ. ID. NO. 9 or one of its        fragments,

said isolated polynucleotide being such that it encodes an isolatedpolypeptide having at least one immunological and/or biological activitycharacteristic of a protein binding human GHRH and being associated withthe modulation of cell proliferation,

with one or more pharmaceutically acceptable excipients.

Preferably, the isolated polynucleotide serving as active ingredientwill consist of the polynucleotide of polynucleotide sequence SEQ. ID.NO. 8 or one of its fragments or the polynucleotide of polynucleotidesequence SEQ. ID. NO. 9 or one of its fragments.

In particular, the isolated polynucleotide serving as active ingredientwill consist of the polynucleotide of polynucleotide sequence SEQ. ID.NO. 8 or the polynucleotide of polynucleotide sequence SEQ. ID. NO. 9.

Said isolated polynucleotide incorporated into a pharmaceuticalcomposition according to the invention is preferably present in a viralvector, said viral vector being for example selected from the groupconsisting of an adenovirus, an associated adenovirus, a retrovirus anda pox virus.

The invention also relates to a pharmaceutical composition comprising anisolated polypeptide comprising a polypeptide of sequence SEQ. ID. NO.14 or one of its fragments, said isolated polypeptide having at leastone immunological and/or biological activity characteristic of a proteinbinding human GHRH and being associated with the modulation of cellproliferation. Preferably, said isolated polypeptide will consist of apolypeptide of sequence SEQ. ID. NO.14 or one of its fragments. Inparticular, said polypeptide will have the sequence SEQ. ID. NO.14.

A subject of the invention is moreover a pharmaceutical compositioncomprising a monoclonal antibody, or an antigen-binding fragment of thelatter, which specifically binds an isolated polypeptide comprising atleast one fragment of the protein encoded by the polynucleotide sequenceSEQ. ID. NO. 9 or by a sequence complementary to the polynucleotidesequence SEQ. ID. NO. 9, said isolated polypeptide having at least oneimmunological and/or biological activity characteristic of a proteinbinding human GHRH and being associated with the modulation of cellproliferation, said composition comprising moreover one or morepharmaceutically acceptable excipients. Preferably, said pharmaceuticalcomposition according to the invention is such that it comprises amonoclonal antibody, or an antigen-binding fragment of the latter, whichspecifically binds an isolated polypeptide consisting of the proteinencoded by the polynucleotide sequence SEQ. ID. NO. 9 or one of thefragments of the latter or by a sequence complementary to thepolynucleotide sequence SEQ. ID. NO. 9 or one of the fragments of thelatter.

In particular, the invention relates to a pharmaceutical compositioncomprising a monoclonal antibody, or an antigen-binding fragment of thelatter, which specifically binds the protein encoded by thepolynucleotide sequence SEQ. ID. NO. 9 or by the sequence complementaryto the polynucleotide sequence SEQ. ID. NO. 9 (in other words theprotein of sequence SEQ. ID. NO. 10) with one or more pharmaceuticallyacceptable excipients.

Another subject of the invention is the use of an isolatedpolynucleotide comprising:

-   -   the polynucleotide sequence SEQ. ID. NO. 8 or one of its        fragments, or    -   the polynucleotide sequence SEQ. ID. NO. 9 or one of its        fragments,

said isolated polynucleotide being such that it encodes an isolatedpolypeptide having at least one immunological and/or biological activitycharacteristic of a protein binding human GHRH and being associated withthe modulation of cell proliferation,

for preparing a medicament intended to treat a proliferative disease.

Preferably, the isolated polynucleotide used will consist of thepolynucleotide of polynucleotide sequence SEQ. ID. NO. 8 or one of itsfragments or the polynucleotide of polynucleotide sequence SEQ. ID. NO.9 or one of its fragments.

In particular, the isolated polynucleotide used will consist of thepolynucleotide of polynucleotide sequence SEQ. ID. NO. 8 or thepolynucleotide of polynucleotide sequence SEQ. ID. NO. 9.

Said isolated polynucleotide used is preferably present in a viralvector, said viral vector being for example selected from the groupconsisting of an adenovirus, an associated adenovirus, a retrovirus anda pox virus.

The present invention also relates to the use of an isolated polypeptidecomprising a polypeptide of sequence SEQ. ID. NO. 14 or one of itsfragments, said isolated polypeptide having at least one immunologicaland/or biological activity characteristic of a protein binding humanGHRH and being associated with the modulation of cell proliferation, forpreparing a medicament intended to treat a proliferative disease.Preferably, said isolated polypeptide will consist of a polypeptide ofsequence SEQ. ID. NO. 14 or one of its fragments. In particular, saidpolypeptide will have the sequence SEQ. ID. NO. 14.

Alternatively, still according to the present invention, a monoclonalantibody, or an antigen-binding fragment of the latter, whichspecifically binds an isolated polypeptide comprising the proteinencoded by the polynucleotide sequence SEQ. ID. NO. 9 or one of thefragments of the latter or by a sequence complementary to thepolynucleotide sequence SEQ. ID. NO. 9 or one of the fragments of thelatter, said isolated polypeptide having at least one immunologicaland/or biological activity characteristic of a protein binding humanGHRH and being associated with the modulation of cell proliferation, canbe used for preparing a medicament intended to treat a proliferativedisease. Preferably, said monoclonal antibody or said fragment of theantigen of the latter will specifically bind a polypeptide consisting ofthe protein encoded by the polynucleotide sequence SEQ. ID. NO. 9 or oneof the fragments of the latter or by a sequence complementary to thepolynucleotide sequence SEQ. ID. NO. 9 or one of the fragments of thelatter.

In particular, a monoclonal antibody, or an antigen-binding fragment ofthe latter, which specifically binds an isolated polypeptide encoded bythe polynucleotide sequence SEQ. ID. NO. 9 or by the sequencecomplementary to the polynucleotide sequence SEQ. ID. NO. 9 (in otherwords, a monoclonal antibody, or an antigen-binding fragment of thelatter, which specifically binds the protein of sequence SEQ. ID. NO.10), can be used for preparing a medicament intended to treat aproliferative disease.

According to preferred variants of the abovementioned uses, theproliferative disease to be treated by the polypeptide or thepolynucleotide described previously is a cancer. According to yet morepreferred variants, the cancer is chosen from the group consisting ofprostate cancer, breast cancer, lung cancer (and in particularsmall-cell lung cancer) and colorectal cancer. Breast cancer andsmall-cell lung cancer are yet more particularly preferred.

The invention moreover offers a method for preparing an isolatedpolypeptide comprising the protein encoded by the polynucleotidesequence SEQ. ID. NO. 9 or SEQ. ID. NO. 13 or one of the fragments ofthe latter or by a sequence complementary to the polynucleotide sequenceSEQ. ID. NO. 9 or one of the fragments of the latter, said isolatedpolypeptide having at least one immunological and/or biological activitycharacteristic of a protein binding human GHRH and being associated withthe modulation of cell proliferation, said preparation method comprisingthe following successive stages:

(a) culture, under suitable conditions in order to obtain the expressionof said polypeptide of a host cell transformed or transfected with anexpression vector comprising an isolated polynucleotide comprising thepolynucleotide sequence SEQ. ID. NO. 9 or SEQ. ID. NO. 13, the sequencecomplementary to the polynucleotide sequence SEQ. ID. NO. 9 or SEQ. ID.NO. 13 or also one of the fragments of the latter, said isolatedpolypeptide having at least one immunological and/or biological activitycharacteristic of a protein binding human GHRH and being associated withthe modulation of cell proliferation, and

(b) isolation of the polypeptide from the host cell cultures.

Preferably, said preparation method will relate to the preparation of anisolated polypeptide consisting of the protein encoded by thepolynucleotide sequence SEQ. ID. NO. 9 or one of the fragments of thelatter or by a sequence complementary to the polynucleotide sequenceSEQ. ID. NO. 9 or one of the fragments of the latter, said isolatedpolypeptide having at least one immunological and/or biological activitycharacteristic of a protein binding human GHRH and being associated withthe modulation of cell proliferation, said preparation method comprisingthe following successive stages:

(a) culture, under suitable conditions in order to obtain the expressionof said polypeptide of a host cell transformed or transfected with anexpression vector comprising an isolated polynucleotide comprising thepolynucleotide sequence SEQ. ID. NO. 9 or SEQ. ID. NO. 13, the sequencecomplementary to the polynucleotide sequence SEQ. ID. NO. 9 or SEQ. ID.NO. 13 or also one of the fragments of the latter, said isolatedpolypeptide having at least one immunological and/or biological activitycharacteristic of a protein binding human GHRH and being associated withthe modulation of cell proliferation, and

(b) isolation of the polypeptide from the host cell cultures.

In particular, the object of said method will be the preparation of theisolated polypeptide encoded by the polynucleotide sequence SEQ. ID. NO.9 or SEQ. ID. NO. 13 or by the sequence complementary to thepolynucleotide sequence SEQ. ID. NO. 9 or SEQ. ID. NO. 13 (in otherwords, the preparation of the protein of sequence SEQ. ID. NO. 10).

According to a yet more preferred variant of said method, the latterwill relate to the preparation of the protein of sequence SEQ. ID. NO.10 and will comprise the following stages:

(a) culture, under suitable conditions in order to obtain the expressionof said polypeptide of a host cell transformed or transfected with anexpression vector comprising an isolated polynucleotide comprising thepolynucleotide sequence SEQ. ID. NO. 9 or SEQ. ID. NO. 13 or thesequence complementary to the polynucleotide sequence SEQ. ID. NO. 9 orSEQ. ID. NO. 13, said isolated polypeptide having at least oneimmunological and/or biological activity characteristic of a proteinbinding human GHRH and being associated with the modulation of cellproliferation, and

(b) isolation of the polypeptide from the host cell cultures.

The present invention also offers a process for identifying compoundscapable of binding human GHRH and modulating cell proliferation, whichcomprises the following successive stages:

(a) bringing each candidate compound into contact with an isolatedpolypeptide comprising:

-   -   either a fragment of the protein encoded by the polynucleotide        sequence SEQ. ID. NO. 9 or by a sequence complementary to the        polynucleotide sequence SEQ. ID. NO. 9,    -   or a fragment of the protein encoded by the polynucleotide        sequence SEQ. ID. NO. 13 or by a sequence complementary to the        polynucleotide sequence SEQ. ID. NO. 13,

under conditions and for a time sufficient to allow the candidate agentto bind to the polypeptide, said isolated polypeptide having at leastone immunological and/or biological activity characteristic of a proteinbinding human GHRH and being associated with the modulation of cellproliferation, and

(b) detection of the binding of each candidate compound to saidpolypeptide and identification, from the candidate compounds, of thecompounds capable of binding human GHRH and modulating cellproliferation.

In particular, said method for the identification of compounds capableof binding human GHRH and modulating cell proliferation will comprise,in its stage (a), bringing each candidate compound, under conditions andfor a time sufficient to allow the candidate agent to bind to thepolypeptide, into contact with the isolated polypeptide encoded by thepolynucleotide sequence SEQ. ID. NO. 9 or by the sequence complementaryto the polynucleotide sequence SEQ. ID. NO. 9 (in other words, with theprotein of sequence SEQ. ID. NO. 10).

An alternative method for the identification of compounds capable ofbinding human GHRH and modulating cell proliferation comprises thefollowing successive stages:

(a) bringing each candidate compound, under conditions and for a timesufficient to allow the candidate agent and the cell to interact, intocontact with a cell capable of expressing an isolated polypeptidecomprising:

-   -   either a fragment of the protein encoded by the polynucleotide        sequence SEQ. ID. NO. 9 or by a sequence complementary to the        polynucleotide sequence SEQ. ID. NO. 9,    -   or a fragment of the protein encoded by the polynucleotide        sequence SEQ. ID. NO. 13 or by a sequence complementary to the        polynucleotide sequence SEQ. ID. NO. 13,

said isolated polypeptide having at least one immunological and/orbiological activity characteristic of a protein binding human GHRH andmodulating cell proliferation, and

(b) determination of the effect of each candidate compound on the cellconcentration of polypeptide and identification, from the candidatecompounds, of the compounds capable of binding human GHRH and modulatingcell proliferation.

In particular, said alternative method will comprise, in its stage (a),bringing each candidate compound, under conditions and for a timesufficient to allow the candidate agent and the cell to interact, intocontact with a cell capable of expressing the isolated polypeptideencoded by the polynucleotide sequence SEQ. ID. NO. 9 or by the sequencecomplementary to the polynucleotide sequence SEQ. ID. NO. 9 (in otherwords, a cell capable of expressing the protein of sequence SEQ. ID. NO.10).

According to preferred methods of implementing the processes foridentifying compounds capable of binding human GHRH and modulating cellproliferation described above, the candidate compounds will originatefrom small molecule libraries resulting from combinatorial chemistryprogrammes.

The pharmacological properties obtained for the polynucleotides andpolypeptides according to the invention make the latter suitable forpharmaceutical use. In fact, the isolated polypeptides comprising:

-   -   either a fragment of the protein encoded by the polynucleotide        sequence SEQ. ID. NO. 9 or by a sequence complementary to the        polynucleotide sequence SEQ. ID. NO. 9,    -   or a fragment of the protein encoded by the polynucleotide        sequence SEQ. ID. NO. 13 or by a sequence complementary to the        polynucleotide sequence SEQ. ID. NO. 13,

which have at least one immunological and/or biological activitycharacteristic of a protein binding human GHRH and being associated withthe modulation of cell proliferation, as well as the polynucleotidesencoding for said polypeptides, can, according to the invention, beadministered to cancer patients in order to slow down the progression oftheir tumors or to make said tumors regress.

In the above methods, the protein binding human GHRH and beingassociated with the modulation of cell proliferation can in particularbe the isolated polypeptide encoded by the polynucleotide sequence SEQ.ID. NO. 9 or by the sequence complementary to the polynucleotidesequence SEQ. ID. NO. 9 (in other words, the protein of sequence SEQ.ID. NO. 10).

Finally, the invention relates to the polynucleotides of sequence SEQ.ID. NO. 4, SEQ. ID. NO. 5, SEQ. ID. NO. 11 and SEQ. ID. NO. 12, whichcan in particular be used as primer in the PCR reactions ofheterocarpine cloning.

The different elements mentioned above will become evident to a personskilled in the art having once read the more detailed description of thedifferent features of the invention.

DETAILED DESCRIPTION OF THE DIFFERENT FEATURES OF THE INVENTION

As mentioned above, the present invention is generally directed towardsproducts and methods intended to modulate cell growth and to treatcancer. The present invention is based, in part, on the identificationof “sequences associated with the modulation of cell proliferation”which are polypeptide and polynucleotide sequences associated with themodulation of cell proliferation. Such cDNA molecules can be preparedfrom preparations of RNA or mRNA using standard techniques, such asreverse transcription. Similarly, a protein or a polypeptide associatedwith differentiation comprises the sequence encoded by an mRNAassociated with cell differentiation.

The pharmaceutical compositions described here can include one or morepolypeptides, nucleic acid sequences and/or antibodies. The polypeptidesof the present invention include at least one portion of the protein ora variant of the latter binding human GHRH and being associated with themodulation of cell proliferation. The nucleic acid sequences of thepresent invention include a DNA or RNA sequence which encodes at leastfor a portion of such a polypeptide or which is complementary to such anencoding sequence.

The antibodies are proteins of the immune system or fragments binding tothe antigen of the latter, which are capable of binding a portion of thepolypeptides described above.

Polynucleotides, Polypeptides and Proteins According to the Invention:

A particular subject of the present invention is the polynucleotides ofsequence SEQ. ID. NO. 8, SEQ. ID. NO. 9 or SEQ. ID. NO. 13 as well asthe polypeptide or the protein of sequence SEQ. ID. NO. 14.

The invention also comprises the polynucleotides having polynucleotidesequences at least 75%, preferably at least 85% and yet morepreferentially at least 90% or even 95%, homologous to the sequences ofthe polynucleotides described above, in particular to the sequences SEQ.ID. NO. 8, SEQ. ID. NO. 9 and SEQ. ID. NO. 13. This also applies mutatismutandis to the other polynucleotides, polypeptides and proteins formingpart of the invention, and in particular to the protein of sequence SEQ.ID. NO. 14.

The degree of homology expressed in % is calculated as follows:100−100×(N′/N)

with N′ representing the number of nucleotides or amino acids modifiedwith respect to the sequence SEQ. ID. NO. 8, SEQ. ID. NO. 9, SEQ. ID.NO. 10, SEQ. ID. NO. 13 or SEQ. ID. NO. 14 and N the number ofnucleotides of the sequence SEQ. ID. NO. 8, SEQ. ID. NO. 9, SEQ. ID. NO.10, SEQ. ID. NO. 13 or SEQ. ID. NO. 14.

According to the invention, the polynucleotide sequences which encodethe polypeptides or proteins of the invention, and fragments or fusionproteins of these polypeptides or proteins, can be used in order togenerate recombinant DNA molecules which direct the expression of thesepolypeptides or proteins, or an active portion of the latter, inappropriate host cells. Alternatively, polynucleotide sequences whichhybridize with portions of the polynucleotide sequences according to theinvention can also be used in nucleic acid hybridization, Southern blot,Northern blot tests, etc.

Because of the degeneration of the genetic encode, other DNA sequencessubstantially encoding for the amino acid sequence of the polypeptidesor proteins of the invention can be used for the cloning and expressionof said polypeptides or proteins. Such DNA sequences include thosecapable of hybridizing the polynucleotide sequences of thepolynucleotides of the invention under certain stringency conditionswhich can be adjusted in several ways. For example, during polymerasechain reaction (PCR), the temperature at which the primers hybridize tothe matrix or the concentrations of MgCl₂ in the reaction buffer, can beadjusted. During the use of radiolabelled DNA fragments, oroligonucleotides to probe membranes, the stringency can be adjusted bychanging the ionic strengths of the washing solutions or by carefullycontrolling the washing temperature.

Preferentially, such a homologous nucleotide sequence hybridizesspecifically with the sequence complementary to the sequence SEQ. ID.NO. 8, SEQ. ID. NO. 9 or SEQ. ID. NO. 13 under stringent conditions (orconditions of “high” stringency). The parameters defining the conditionsof stringency depend on the temperature at which 50% of the pairedstrands separate (T_(m)).

For the sequences comprising more than 30 bases, and according toSambrook et al.. (Molecular cloning: A Laboratory Manual, Cold SpringHarbor Laboratories, Cold Spring Harbor, N.Y., 1989), T_(m) is definedby the equation:T_(m) = 81.5 + 0.41 × (%  G + C) + 16.6 × log   [cations] − 0.63 × (%  formamide) − (600/number  of    bases)

For the present invention, the stringency conditions of are called“high” when a hybridization temperature 10° C. below T_(m) andhybridization buffers containing a 6×SSC (0.9 M sodium chloride and 0.09M sodium citrate) solution are used. Under such conditions, thepolynucleotides of aspecific sequences will not hybridize with thepolynucleotide of the sequence complementary to the sequence SEQ. ID.NO. 8, SEQ. ID. NO. 9 or SEQ. ID. NO. 13.

Altered DNA sequences which can be used in accordance with the presentinvention include deletions, additions or substitutions of differentnucleotide residues resulting in a sequence which encodes the same geneproduct or equivalent function. The gene product can also containdeletions, additions or substitutions of amino acid residues in thesequences of the proteins of the invention, which result in so-calledsilent changes, thus producing polypeptides and proteins of equivalentfunction.

Such amino acid substitutions can be carried out on the basis of thepolarity, charge, solubility, hydrophobicity, hydrophilicity, and/oramphipatic nature of the residues involved.

For example, negatively-charged amino acids include aspartic acid andglutamic acid, positively-charged amino acids include lysine andarginine, amino acids with polar groups having close hydrophobicityvalues include leucine, isoleucine, valine; glycine, alanine;asparagine, glutamine; serine, threonine; phenylalanine, tyrosine.

The DNA sequences of the present invention can be modified in order toalter the polynucleotide sequences according to the invention fornumerous reasons including in a non-limitative manner alterations whichmodify the process and the expression of the gene product. For example,mutations can be introduced using techniques well known to a personskilled in the art, for example directed mutagenesis, the insertion ofnew restriction sites, the alteration of the glycosylations,phosphorylation, etc.

In particular, in certain expression systems such as yeast, the hostcell can over-glycosylate the gene product. In such a system, it ispreferable to alter the polynucleotide sequences in order to eliminatethe glycosylation sites. Within the scope of the disclosure of thepresent invention modified polynucleotide sequences also figure, linkedwith heterologous sequences in order to encode a fusion protein. Thefusion protein (which can be for example the protein of sequence SEQ.ID. NO. 14) can be modified in order to contain a cleavage site locatedbetween the sequence of the protein according to the invention (forexample the sequence SEQ. ID. NO. 10) and the sequence of theheterologous protein, such that the sequence of the protein according tothe invention can be cleaved from the heterologous part.

Polynucleotides Encoding for Polypeptides Associated With the Modulationof Cell Proliferation:

Any polynucleotide which encodes a polypeptide or a portion or a variantof the latter as described here, binding human GHRH and being associatedwith the modulation of cell proliferation, is covered by the presentinvention. Such polynucleotides can be single-strand (encoding oranti-sense) or double-strand and can be DNA (genomic, cDNA or synthetic)or RNA molecules.

The polynucleotides encoding for polypeptides binding human GHRH andbeing associated with the modulation of cell proliferation can beprepared using any technique available to a person skilled in the art.For example, such a polynucleotide can be amplified via a polymerasechain reaction (PCR) from cDNA prepared from cells. For this approach,specific primers can be designed and ordered or synthesized; theseprimers are based on the sequence of said polynucleotide. An amplifiedportion can then be used in order to isolate the complete gene of anycell or any tissue from a genomic DNA library or from a cDNA library, bymeans of techniques well known to a person skilled in the art andbriefly recalled below. Alternatively, a complete gene can beconstructed from several PCR fragments. The cDNA molecules encoding fora protein binding human GHRH and being associated with the modulation ofcell proliferation, or a portion of the latter, can also be prepared byscreening a cDNA library obtained for example from mRNA of cells ortissues. Such libraries can be commercially available or can be preparedusing the standard techniques (cf. Sambrook et al., Molecular cloning: ALaboratory Manual, Cold Spring Harbor Laboratories, Cold Spring Harbor,N.Y., 1989).

Alternatively, other screening techniques well known to a person skilledin the art can be used.

A cDNA molecule encoding for a polypeptide binding human GHRH and beingassociated with the modulation of cell proliferation can be sequenced byusing the standard techniques using enzymes such as the Klenow fragmentof DNA polymerase I, Sequenase X (US Biochemical Corp., Cleveland, Ohio,United States), Taq polymerase (Perkin Elmer, Foster City, Calif.,United States), thermostable polymerase T7 (Amersham, Chicago, IT.,United States) or a combination of recombinant polymerases andexonucleases with a rereading activity such as the Elongaseamplification system (Gibco BRL, Gaithersburg, Md., United States). Anautomatic sequencing system can be used, using instruments availablefrom commercial suppliers such as Perkin Elmer and Pharmacia.

The partial sequence of a cDNA can be used in order to identify apolynucleotide sequence which encodes the complete protein associatedwith the modulation of cell proliferation using standard techniques wellknown to a person skilled in the art. Among these techniques, a cDNAlibrary is screened using one or more polynucleotide probes using RecArecombination properties (ClonCapture cDNA Selection Kit, ClontechLaboratories, United States).

For the hybridization techniques, a partial sequence can beradiolabelled (for example by nick translation or labelling of the endsusing ³²P or ³³P) using standard techniques. A bacteria or bacteriophagelibrary is then screened by hybridization on filters containingdenatured bacterial colonies (or blots containing phage plates) with thelabelled probe (cf. Sambrook et al., Molecular cloning: A LaboratoryManual, Cold Spring Harbor Laboratories, Cold Spring Harbor, N.Y.,1989). The positive colonies or plates are then selected and amplifiedand the DNA is isolated for future analyses.

The complete sequence can then be determined using standard techniques.The overlapping sequences are then assembled into a continuous singlesequence. A complete cDNA molecule can be generated by ligation of thefragments of interest using standard techniques.

Alternatively, numerous techniques based on amplification exist forobtaining a complete encoding sequence from a partial cDNA sequence.Among these, the amplification is generally carried out via PCR. All ofthe kits commercially available can be used for the amplificationstages. The primers can be designed using, for example, software wellknown in the art. The nucleotide primers are preferably molecules with20 to 30 nucleotides having a guanine and cytosine content of at least50% and which hybridize with the target sequence at temperaturescomprised between 50 and 72° C. The amplified region can be sequenced asdescribed above and the overlapping sequences assembled into acontinuous sequence.

Among the alternative approaches, sequences adjacent to the partialsequence can be found by amplification with a primer of the bindingsequence and a primer specific to a known region. The amplifiedsequences are then subjected to a second amplification cycle.

Additional techniques include capture PCR (Lagerstrom et al., PCRMethods Applic. (1991), 1, 111-19) and progressive PCR (Parker et al.,Nucl. Acids. Res. (1991), 19, 3055-60). Other methods usingamplification can also be used in order to obtain a complete cDNAsequence.

It is possible to obtain a complete cDNA sequence by analyzing thesequences deposited in the public “Expressed Sequence Tags” (ESTs) basesavailable from GenBank. Research covering the ESTs can be carried outusing computer programmes well known to a person skilled in the art (forexample NCBI BLAST) and such ESTs can be used to generate a continuouscomplete sequence.

Variants of the polynucleotide sequences described above (in particularsequences SEQ. ID. NO. 8, SEQ. ID. NO. 9 and SEQ. ID. NO. 13) are alsoincluded within the scope of the present invention. The polynucleotidevariants can contain one or more substitutions, deletions or insertions(cf. also above in the part entitled “Polynucleotides, polypeptides andproteins according to the invention”).

A portion of the sequence complementary to the encoding sequence (i.e.an anti-sense polynucleotide) can also be used as a probe or modulatorof gene expression. The cDNA construct which can be transcribed toanti-sense RNA can be introduced into cells or tissues in order tofacilitate the production of anti-sense RNA. An anti-sensepolynucleotide can be used, as described here, in order to inhibit theexpression of a gene associated with the modulation of cellproliferation. The anti-sense technology can be used in order to controlgene expression by forming a triple-helix, which compromises the abilityof the double helix to open sufficiently for the fixing of polymerases,transcription factors or regulation molecules (cf. Gee et al. in Huberand Carr, Molecular and Immunologic Approaches (1994), Futura PublishingCo., Mt. Kisco, N.Y.). Alternatively, an anti-sense molecule can be usedto hybridize with a gene control region (for example a promoter orinitiation site of the transcription) and to block the transcription ofthe gene, or to block the translation by inhibiting the fixing ofribosomes to the transcript.

The polynucleotides can then be modified in order to increase theirstability in vivo. Possible modifications include (but are not limitedto): the addition of sequences to the 5′ and/or 3′ ends; the use ofphosphorothioate or 2′ O-methyl rather than phosphodiesterase bonds inthe backbone; and/or the introduction of bases such as inosine, queosineand wybutosine as well as acetyladenine, methylthioadenine and modifiedforms of adenine, cytidine, guanine, thymine and uridine.

Other variations of the polynucleotides of the present invention havemoreover already been described previously in the part entitled“Polynucleotides, polypeptides and proteins according to the invention”.

The nucleotide sequences as described in the present invention can bejoined to other nucleotide sequences using established recombinant DNAtechniques. For example, a polynucleotide can be cloned in a large panelof expression vectors, including plasmids, phagemids, lambda phagederivatives and cosmids. The vectors of particular interest include theexpression vectors, replication vectors and sequencing vectors. Ingeneral, a vector contains a functional replication origin in at leastone organism, suitable endonuclease restriction sites and one or moreselection markers. The presence of other elements will depend on thespecific use desired by a person skilled in the art who will select thecharacteristics of the expression vector as a function of hisrequirements and the techniques available.

The polynucleotides can be formulated in order to enter the cell andexpress the corresponding polypeptide. Such formulations areparticularly useful in therapy as described hereafter.

Persons skilled in the art will appreciate that several means exist forexpressing a polynucleotide in a target cell, and that any appropriatetechnique can be used. For example, a polynucleotide can be incorporatedinto a viral vector such as an adenovirus or a retrovirus (but also intoothers). Techniques for incorporating DNA into such vectors are wellknown to a person skilled in the art. A retroviral vector can transferor incorporate a gene for a selection marker and/or a screening entitysuch as a gene encoding for the ligand of a specific receptor of atarget cell, in order to render the vector target-specific.

Other formulations for the polynucleotides include colloidal dispersionsystems such as macromolecular complexes, nano-capsules, microspheres,spheres, and systems based on the use of lipids including oil/wateremulsions, micelles, mixed micelles and liposomes. The preferredcolloidal system for use for delivering the product in vitro and in vivois the liposome (i.e. an artificial membrane vesicle).

Polypeptides Binding Human GHRH and Modulating Cell Proliferation:

Within the scope of the disclosure, the polypeptides of the presentinvention include at least one portion of the protein associated withthe modulation of cell proliferation or a variant of the latter, saidportion being immunologically and/or biologically active. Suchpolypeptides can have any length, including the complete protein, anoligopeptide (i.e. consisting of a relatively limited number of aminoacids, such as 8-10 residues, joined by peptide bonds) or a peptide ofintermediate size. A polypeptide can also comprise additional sequences.

Similarly, a polypeptide is “biologically active” if it has one or morestructural, regulatory and/or biochemical functions starting with thenative protein associated with the binding of human GHRH and beingassociated with the modulation of cell proliferation.

The presence of a biological activity can be determined according toprocesses well known to a person skilled in the art. However, bydefinition within the framework of the present invention, a polypeptideis considered as “having at least one immunological and/or biologicalactivity characteristic of a protein binding human GHRH and beingassociated with the modulation of cell proliferation” once itsinhibitory concentration IC₅₀ measured under the conditions described inExample 6 of the present Application is less than or equal to 10 nM (andpreferably less than or equal to 1 nM).

For example, comparative studies of sequences can indicate a particularbiological activity of the protein. Tests for evaluating said activitycan then implemented on the basis of tests already known in the art.Certain portions and other variants of such proteins should also showthis property according to an in vitro or in vivo test.

As already mentioned, the polypeptides according to the presentinvention can comprise one or more portions of a variant of theendogenous protein where the portion is immunologically and/orbiologically active (i.e. the portion has one or more antigenic,immunogenic and/or biological characteristics of the complete protein).Preferably, such a portion is at least as active as the total proteinduring tests allowing the detection of such properties. A “variant”polypeptide is a polypeptide which differs from the native protein bysubstitutions, insertions, deletions and/or modifications of aminoacids. Certain variants contain conservative substitutions. “Aconservative substitution” is a substitution in which one amino acid issubstituted by another amino acid having the same properties, such asthose determined by a person skilled in the art who expects no change inthe secondary structure, as well as in the hydropathic nature of thepolypeptide. The amino acid substitutions can generally be carried outon the basis of similarity of polarity, charge, solubility,hydrophobicity, hydrophilicity, and/or amphipathic nature of theresidues. For example, negatively-charged amino acids include asparticacid and glutamic acid; positively-charged amino acids include lysineand arginine; and polar non-charged amino acids having similarhydrophobicity values include leucine, isoleucine and valine; glycineand alanine; asparagine and glutamine; serine, threonine, phenylalanineand tyrosine. Other amino acid groups which can represent conservativechanges are in particular the following: (1) Ala, Pro, Gly, Glu, Asp,Gln, Asn, Ser, Thr; (2) Cys, Ser, Tyr, Thr (3) Val, Ile, Leu, Met, Ala,Phe; (4) Lys, Arg, His; and (5) Phe, Tyr, Trp, His. A variant can also,or alternatively, contain non-conservative changes.

Variants forming part of this invention also include polypeptides inwhich the primary structure of the native protein is modified byformation of covalent conjugates or not modified with other polypeptidesor chemical structures such as lipid groups or glycosyl, or phosphateacetyl groups.

The present invention also includes polypeptides with or withoutglycosylation patterns. The polypeptides expressed in expression systemsof yeast or mammal cells can, in terms of molecular weight andglycosylation pattern, be similar to or slightly different from thenative molecule according to the expression system used.

The expression of DNA in bacteria such as E. Coli leads tonon-glycosylated molecules. The N-glycosylation sites of the eucaryoticproteins are characterized by the triplet of amino acids Asn-A1-Z whereA1 is any amino acid except Pro, and Z is a serine or a threonine.

Other variations of the polypeptides and proteins of the presentinvention have moreover already been described previously in the partentitled “Polypeptides and polynucleotides according to the invention”.

In order to prepare a polypeptide variant, standard mutagenesistechniques, such as directed mutagenesis using a directedoligonucleotide, can be used.

In general, any expression vector known to a person skilled in the artcan be used in order to express recombinant polypeptides of thisinvention. The expression can be obtained in any appropriate host cellwhich has been transformned or transfected with an expression vectorcontaining a DNA sequence which encodes the recombinant polypeptide.Suitable host cells include procaryotic, higher eucaryotic or yeastcells. Preferably, the host cells used are E. Coli, yeast cells ormammal cells such as COS, CHO, HEK-293, MCF7 (human tumor cells isolatedfrom a mammary carcinoma) or DU 145 (human tumor cells isolated from aprostate cancer).

Certain portions and other variants can also be generated by syntheticmeans using techniques well known to a person skilled in the art. Forexample, portions and other variants having less than 500 amino acids,preferably less than 100 amino acids and more preferentially less than50 amino acids can be synthesized by chemical route. The polypeptidescan be synthesized using solid phase synthesis techniques availablecommercially, such as the Merrifield resin synthesis method where theamino acids are sequentially added to a chain of amino acids during thesynthesis process (cf. Merrifield, J. Am. Chem. Soc. (1963), 85,2149-2146). Numerous other solid phase synthesis techniques are alsoavailable (for example the process of Roberge et al., Science (1995),269, 202-204). Equipment for the automatic synthesis of polypeptides iscommercially available from suppliers such as Applied Biosystems, Inc.(Foster City, Calif., United States); the synthesis of the polypeptidescan then be carried out by following the manufacturer's recommendations.

Isolated Polynucleotides or Polypeptides:

In general, the polypeptides and polynucleotides described in thepresent invention are isolated. An “isolated” polypeptide orpolynucleotide is a polynucleotide or a peptide removed from itsoriginal environment. For example, a natural protein is isolated if itis separated from the biological material with which it coexists in thenatural system. A polynucleotide is considered as isolated if, forexample, it is cloned in a vector which does not form part of thenatural environment.

Antibodies and Fragments of the Latter:

The present invention provides binding agents, such as the antibodieswhich specifically bind the protein associated with the binding of humanGHRH and being associated with the modulation of cell proliferation.Such an agent is referred to as “specifically binding” to the cellproliferation modulation protein if it reacts at a level which isdetectable (for example by an ELISA test) with a protein associated withthe modulation of cell proliferation or a portion or a variant of thelatter and does not react in detectable manner with other proteins. “Thebinding” refers to a non-covalent association between 2 separatemolecules such that a complex is formed. The binding ability can beevaluated, for example, by determination of the binding constant for theformation of the complex. The binding constant is the value obtainedwhen the value of the concentration of the complex is divided by theproduct of the values of the concentration of the components. Ingeneral, 2 products are called “bound” when the binding constant reaches103 l/mol. The binding constant can be determined using methods wellknown to a person skilled in the art.

Any agent capable of fulfilling the above criteria can be considered asa binding agent.

In the present invention, a binding agent is preferably an antibody or afragment of the latter. The antibodies can be prepared by any techniqueavailable to a person skilled in the art (cf. Harlow and Lane,Antibodies. A Laboratory Manual, Cold Spring Harbor Laboratory, 1988).In general, the antibodies can be produced by cell culture techniquesincluding the generation of monoclonal antibodies or via transfectionsof genes of antibodies in host cells of bacteria or mammals in order toproduce recombinant antibodies.

Among other techniques, it is preferable to use those describedhereafter. An immunogen containing the polypeptide is injected into agroup of mammals (for example mice, rats, rabbits, sheep or goats). Inthis stage, the polypeptides of the present invention can serve asimmunogens without modification. Alternatively, and particularly forsmall peptides, a better immune response can be induced if thepolypeptide is joined to a transport protein such as bovine serumalbumin or keyhole-limpet hemocyanin. The immunogen is injected into thehost animal, preferably according to a predetermined schema, and theanimals are bled periodically. Polyclonal antibodies specific to thepolypeptide can thus be purified from such antisera, for example byaffinity chromatography using the peptide coupled to an appropriatesolid support.

In order to prepare an antibody specifically binding a protein ofsequence A but not a protein of sequence B, the protein of sequence A isinjected into the host animal, preferably according to a predeterminedschema, and the animal are bled periodically. Polyclonal antibodiesspecific to the polypeptide of sequence A can thus be purified from suchantisera, for example by affinity chromatography using the protein ofsequence B coupled to an appropriate solid support. The correspondingeluate contains the antibody specifically binding a protein of sequenceA but not a protein of sequence B.

Fusion Proteins:

Any fusion gene can be produced by a person skilled in the art in orderto analyze the sub-cellular location of a protein according to theinvention, in particular the sub-cellular location of the protein ofsequence SEQ. ID. NO. 10. Numerous plasmidic constructions are availablecommercially such as Glutathione S Transferase (GST) protein orfluorescent proteins such as Green Fluorescent Protein (GFP) or also andnon-exhaustively a polyhistidine tag.

Human eucaryotic host cells (for example HEK-293) are sub-cultured for24 hours before the transfection protocol allowing normal metabolism ofthe cells and better transfection effectiveness. Increasingconcentrations (1, 5 and 10 μg) of vector alone containing the developerprotein (GFP, GST or Tag Histidine) or of vector containing thepolynucleotide of sequence SEQ. ID. NO. 8 or the polynucleotide ofsequence SEQ. ID. NO. 9 fused with the developer protein were producedusing the reagent Effectene® according to the manufacturer's (Qiagen)recommendations.

The cells are then analyzed by confocal microscopy, for example, inorder to detect the location of the protein. If the protein is suspectedof being secreted for example, the supernatants are recovered,lyophilized, deposited on acrylamide gel and analyzed by the Westernblot technique using antibodies directed against the developer protein.

Pharmaceutical Compositions:

According to certain features of the invention, products such aspolypeptides, antibodies and/or nucleic acids can be incorporated intopharmaceutical compositions or vaccines. The pharmaceutical compositionscomprise one or more of these products and one or more pharmaceuticallyacceptable excipients (carriers). Certain pharmaceutical compositionsoptionally usable as vaccines can comprise one or more polypeptides andan immune response activator, such as an adjuvant or liposome (intowhich the product is incorporated). The pharmaceutical compositions andthe vaccines can moreover contain an administration system, such asbiodegradable microspheres (and for example microspheres composed oflactic acid and glycolic acid copolymers or PLGA). The pharmaceuticalcompositions and the vaccines within the scope of the disclosure of thepresent invention can also contain other products being able to bebiologically active or inactive.

A pharmaceutical composition or a vaccine can contain DNA encoding forone or more polypeptides as described above, such that the polypeptideis generated in situ. As mentioned previously, the DNA can be present inany administration form known to a person skilled in the art, includingbacterial or viral expression systems of nucleic acids. The appropriateexpression systems of nucleic acids contain the DNA sequences necessaryfor expression in the patient.

The administration systems based on a bacterium involve theadministration of a bacterium (such as Bacillus-Calmette-Guerrin) whichexpresses an immunogenic portion of the polypeptide at its surface.Preferably, the DNA can be introduced using a viral expression system(for example a pox virus, retrovirus or adenovirus) involving the use of(defective) non-pathogenic agents.

Although any appropriate carrier known to a person skilled in the artcan be used in pharmaceutical compositions of this invention, the typeof carrier will vary according to the chosen administration method. Thecompositions of the present invention can be formulated for eachappropriate administration method, including, for example, the topical,nasal, intravenous, intra -cranial, intra-peritoneal, sub-cutaneous andintramuscular routes.

For a parenteral administration, such as a sub-cutaneous injection, thecarrier preferably contains water, salt, alcohol, fat, paraffin or abuffer. For oral administration, any carrier mentioned above or a solidcarrier, such as mannitol, lactose, starch, magnesium stearate, talc,cellulose, glucose, sucrose and magnesium carbonate can be used.Biodegradable microspheres can also be used as carriers for thepharmaceutical compositions of this invention. For certain topicalapplications, formulations such as creams or lotions are preferred.

Such compositions can also comprise buffers (for example neutral orphosphate buffered saline solutions), carbohydrates (for exampleglucose, mannose, sucrose or dextrans), mannitol, proteins, polypeptidesor amino acids such as glycine, antioxidants, chelating agents such asEDTA or glutathione, adjuvants (for example aluminium hydroxide) and/orprotective agents. Alternatively, the compositions of the presentinvention can be presented in the form of a lyophilisate. Products canalso be encapsulated in liposomes using standard technologies.

According to the invention, each of the varieties of adjuvant can beused in vaccines in order to induce the immune response. Most of theadjuvants contain a substance protecting the antigen from a rapidcatabolism, such as aluminium hydroxide or mineral oil and an immuneresponse stimulator such as lipid A, proteins derived from BordetellaPertussis or Mycobacteium tuberculosis. Appropriate adjuvants arecommercially available such as, for example: Freund's adjuvant andcomplete adjuvant (Difco Laboratories, Detroit, MIY., United States;Merck Adjuvant 65 (Merck and Company, Inc., Rahway, N.J., UnitedStates)), biodegradable microspheres; monophosphoryl lipid A; andcytokines such as GM-CSF or interleukin-2, -7 or -12.

The compositions described above can also be administered in the form ofretard formulations (i.e. a formulation such as a capsule or a spongewhich triggers the slow release of the product after administration).Such formulations can generally be prepared using technologies wellknown to a person skilled in the art and administered, for example, byoral, rectal route or sub-cutaneous implantation or implantation at thedesired target site. The retard formulations can contain a polypeptide,a polynucleotide or an antibody dispersed in a carrier matrix and/orcontained in a reservoir protected by a diffusion membrane. The carriersfor the use of such formulations are biocompatible and must also bebiodegradable; preferably the formulation provides a relatively constantlevel of release of the active component. The quantity of active productcontained in the retard formulation depends on the implantation site.

Anticancer Therapy

According to other features of the present invention, the productsdescribed can be used in anticancer therapy. In particular, thepolynucleotides and polypeptides associated with the binding of humanGHRH and being associated with the modulation of cell proliferation canbe used in order to inhibit growth and induce the modulation of cellproliferation in tumours specific to the breast, the prostate or lungcancer.

Such polypeptides or polynucleotides can also be used for the therapy ofnumerous carcinomas including melanomas, the multiple forms ofglioblastomas, carcinomas of the lung as well as colorectal cancers.Agents which activate the expression of such polypeptides orpolynucleotides can also be used within the framework of thesetherapies.

According to these aspects of the invention, the products (which can bepolypeptides or nucleic acids) are preferably incorporated intopharmaceutical compositions as described above.

Appropriate patients for the therapy are all warm-blooded animals, andpreferably human beings. A patient eligible for a therapy according tothe invention may or may not be diagnosed as affected by a cancer. Inother words, the pharmaceutical compositions described above can thus beused in order to inhibit the development of a cancer at different stagesof the disease (in order to prevent the appearance of a cancer or inorder to treat a patient affected by a cancer).

The pharmaceutical compositions of the present invention areadministered in a manner appropriate to each specific cancer to betreated.

The route, duration and frequency of administration are determined as afunction of the state of the patient, type and severity of the disease,and administration method. The routes and frequencies of administrationcan vary from one individual to another. In general, the pharmaceuticalcompositions and the vaccines can be administered by injection (forexample by intra-cutaneous, intramuscular, intravenous or sub-cutaneousroute), by intra-nasal route (for example by inhalation) or by oralroute. Preferably, between 1 and 10 doses can be administered over a52-week period. Alternative protocols may be appropriate for eachpatient individually.

In general, an appropriate dosage and a treatment regime contains theactive ingredient in a quantity sufficient to provide a therapeuticand/or prophylactic benefit. Such a response can be followed by theestablishment of an improved clinical outlook (for example more frequentremissions, survival in the complete, partial or longer absence of thedisease) in patients treated compared with patients not treated ortreated with smaller doses.

According to other features of the present invention, a polypeptide canbe administered in doses varying from 100 μg to 5 mg. The DNA moleculesencoding for such polypeptides can generally be administered in aquantity sufficient to generate comparable polypeptide levels.Appropriate dosages can generally be determined using experimentalmodels and/or clinical tests. In general, the use of the minimum dosesufficient to provide an effective therapy is preferred. The patientscan generally be monitored with regard to the effectiveness of thetherapy using tests appropriate to the conditions of treatment orprevention which will appear familiar to a person skilled in the art.

Unless otherwise defined, all the technical and scientific terms usedhere have the same meaning as that commonly understood by an ordinaryspecialist in the field to which this invention belongs. Similarly, allthe publications, patent applications, all the patents and all otherreferences mentioned here are incorporated by way of reference.

The following examples are presented in order to illustrate the aboveprocedures and should in no case be considered as a limit to the scopeof the invention.

EXAMPLES Example 1 Cloning of the cDNA Encoding for Heterocarpine

1.1 ) Extraction of the RNAs from Pilocarpus Heterophyllus Cells:

The cells in culture are preserved at −80° C. before the stages of totalRNA extraction. The total RNA extraction is based on a techniquedescribed in the scientific literature (Chomczynski and Sacchi, Anal.Biochem. (1987), 162, 156) using Trizol reagent (Gibco/BRL). The qualityof the RNAs thus extracted is analyzed on 1% agarose gel in the presenceof ethidium bromide.

1.2) Synthesis of the cDNAs By Reverse Transcription:

The RNAs are retrotranscribed according to two different operatingmethods in order to dissociate and promote the reverse transcriptions ofthe 5′ and 3′ parts of the RNAs using the SMART™ RACE cDNA AmplificationKit (Clontech).

1.3) Design and Synthesis of Primers for Polymerase Chain Reaction(PCR):

The amplification of the 2 sequences specific to the cDNA ofheterocarpine was carried out by polymerase chain reaction (PCR) onreverse transcription products using the Rev1 primer for the products of5′ specific cDNA and the Fwd1 primer for the products of 3′ specificcDNA of respective sequences SEQ. ID. NO. 4 and SEQ. ID. NO. 5.

The sequences SEQ. ID. NO. 4 and SEQ. ID. NO. 5 are the following: SEQ.ID. NO.4: 5′-TCC AAG CAG CAA AAA CTA GTG ACC CAG GGG CCA TTA TAT CT-3′SEQ. ID. NO.5: 5′-CGG TAT GGA CGC GGC TAT TGC TGA TGG TGT TGA TGT AA-3′

1.4) Polymerase Chain Reaction (PCR) and Results:

The reaction conditions include 0.2 μM of Fwd1 for the products of 3′specific cDNA and 0.2 μM of Rev1 for the products of 5′ specific cDNA,200 μM dNTPs, 40 mM Tricine-KOH (pH 8.7), 15 mM KOAc, 3.5 mM Mg(OAc)₂,3.75 μg/ml BSA, 0.005% Tween-20, 0.005% Nonidet-P40, and 0.5 U Taq DNApolymerase in a final volume of 50 μl. The PCR reactions are carried outin a Perkin-Elmer 9700 thermocycler using the following thermal cycleparameters: 5 cycles comprising a denaturation at 94° C. for 5 seconds,a hybridization of the primers at 72° C., 5 cycles comprising adenaturation at 94° C. for 5 seconds, a hybridization of the primers at70° C. for 10 seconds, and an extension of polymerization at 72° C. for3 minutes and finally 25 cycles comprising a denaturation at 94° C. for5 seconds, a hybridization of the primers at 68° C. for 10 seconds, anda polymerase extension at 72° C. for 3 minutes.

The products obtained by PCR are separated on 1% agarose gel andvisualized using ethidium bromide staining.

The nucleic acid sequences of the products of 5′ specific and 3′specific cDNA PCR are determined using an automatic sequencer. These arerespectively the sequences SEQ. ID. NO. 6 and SEQ. ID. NO. 7 reproducedhereafter: SEQ. ID. NO.6: 1 ctaatacgac tcactatagg gcaagcagtg gtatcaacgcagagtacgcg gggatgcccc 61 aagctaattc ttatcttttt tctttctttt tgttgttgttttgtcaaagc agcaatgagg 121 tctaggaatg gtgttcttca tttattcctt ttcgttcttgcatggcttct gttggcggct 181 ctccatgcta actcaagttc ggatgagaga tcaacatatatagttcatat ggacaagacc 241 catatgccca aaaccttctc tagcccccac cattggtactcttcggtcgt tcgatccctc 301 aagtctacaa agccaaccaa attaaatcgc cgtcgatcctcaccacttct tgtatactct 361 tacgacaatg ctgctcatgg tttcagtgca gttttatctcaacaggaact tgaaactcta 421 aaaaagtctc caggtttcgt ctcagtttat gccgataagacagcgacact tgacaccacc 481 catacacctg aatttctctc cctgaatact gccaacgggttgtggcctgc ttcaaagtat 541 ggtgaagata taattgttgg tgttattgac agcggtgtctggccggagag tgaaagttat 601 aatgatgatg gtatgggcgc tattccaagc agatggaagggagaatgtga agctggacaa 661 gagttcaatt cctccatgtg caactcaaag cttattggagctagatattt cgataagggt 721 atcattgcgg caaatcctgg gattaacatt agcatgaaatctgccagaga tactatgggg 781 catgggactc acacatcctc cacagttgct gggaattatgtggatggcgt ttcattcttt 841 ggctatgcta aaggtacagc aaaaggagtg gcaccacgggcgagagtggc tatgtacaag 901 gtcatttttg acgaagggcg ctatgcatct gatgttcttgccggtatgga cgcggctatt 961 gctgatggtg ttgatgtaat ttcaatatca atgggatttgatgagacccc gttgtatgaa 1021 gatcctatag caattgcctc attcgctgct acagagaagggcgtagtggt ctcatcttca 1081 gcaggaaatg cagggccagc gctagggagc ttgcacaatggaatcccatg gacgttaact 1141 gttgcagctg gaaccattga ccgttcattt gcaggcactataactcttgg gagtggggaa 1201 accatcattg gatggacaat gttcccagcc agtgcttatgtagcagactt gccactgctt 1261 tataacaaga cttactctgc atgcaactca actcgattattatctcaact ccgaactgac 1321 gccatcatcg tatgcgaaga agctgaagat tcggtatctgagcaaatatc tgttgtcagt 1381 gcatcgaaca ttcggggagc catatttgtt tcagattatgatgctgaatt atttgaactt 1441 ggtggtgtga ctattcctgg tgtcgtgatt agcaccaaggatgcaccggc tgtgatcagc 1501 tacgccagca atgatgtgaa acctaaggca agcatcaagttccaacaaac tgttctgggc 1561 acaaagcctg caccagccgt ggctttctat acttctagaggtccgtcacc gagctatcca 1621 ggcatcttaa agccagatat aatggcccct gggtcactagtttttgctgc ttgga SEQ. ID. NO.7: 1 cggtatggac gcggctattg ctgatggtgttgatgtaatt tcaatatcaa tgggatttga 61 tgagaccccg ttgtatgaag atcctatagcaattgcctca ttcgctgcta cagagaaggg 121 cgtagtggtc tcatcttcag caggaaatgcagggccagcg ctagggagct tgcacaatgg 181 aatcccatgg acgttaactg ttgcagctggaaccattgac cgttcatttg caggcactat 241 aactcttggg agtggggaaa ccatcattggatggacaatg ttcccagcca gtgcttatgt 301 agcagacttg ccactgcttt ataacaagacttactctgca tgcaactcaa ctcgattatt 361 atctcaactc cgaactgacg ccatcatcgtatgcgaagaa gctgaagatt cggtatctga 421 gcaaatatct gttgtcagtg catcgaacattcggggagcc atatttgttt cagattatga 481 tgctgaatta tttgaacttg gtggtgtgactattcctggt gtcgtgatta gcaccaagga 541 tgcaccggct gtgatcagct acgccagcaatgatgtgaaa cctaaggcaa gcatcaagtt 601 ccaacaaact gttctgggca caaagcctgcaccagccgtg gctttctata cttctagagg 661 tccgtcaccg agctatccag gcatcttaaagccagatata atggcccctg ggtcactagt 721 ttttgctgct tggattccaa atactgctacagcccaaatt ggtttgaata ccctcttgac 781 aagtgaatac aatatggttt ctggaacatcaatggcctgc cctcatgctg ctggtgtagc 841 tgctctcctt aagggcgcac accctgaatggagtgcagct gctattaggt ctgcaatgat 901 gactacagca aatcccttgg ataacacactaaatccaatc cgggacaatg gtctaatcaa 961 tttcacatct gcttcacctt tagctatgggagccggccaa gttgatccta atcgggcact 1021 tgatcctggt ttgatttatg aaaccaccccacaagattat gtgagcctcc tctgcactct 1081 gaacttcacc caaaaccaaa tcctgtccattacaagatca aaccgttaca gctgctccac 1141 ccctaatcct gatcttaact atccttcttttattacttta cactacaaca caaatgcaac 1201 atttgttcag acttttcaca ggactgtgactaacgttgga ggaagcgcta caacttacaa 1261 ggccaagatc actgctcctc taggttctgtagttagtgtc tcaccagaca cattggcctt 1321 cagaaagcag tatgagcagc agagctacqagctcactatt gagtacaagc ctgatggtga 1381 agaaactgtt tcatttgggg aacttgtttggattgaagaa aatgggaatc acactgtgag 1441 gagccctatt acagtgtcac cttccatgagtaactttgtg tttatgggta cacaataatt 1501 gataaaaatt tgttctgatc acaactgtgggaataatcga cgtttatgaa cccagaataa 1561 gttgtttggt cgtcttcaac attatcataaaggacttgaa tcatgtgtgt tgattttctg 1621 caaaaaaaaa aaaaaaaaaa aagtactctgcgttgatacc actgcttgcc ctatagtgag 1681 tcgtattag

The overlapping sequences SEQ. ID. NO. 6 and SEQ. ID. NO. 7 make itpossible to deduce the complete sequence of the cDNA of sequence SEQ.ID. NO.8 encoding for heterocarpine. The sequence SEQ. ID. NO.8 isreproduced hereafter: SEQ. ID. NO.8: 1 ctaatacgac tcactatagg gcaagcagtggtatcaacgc agagtacgcg gggatgcccc 61 aagctaattc ttatcttttt tctttctttttgttgttgtt ttgtcaaagc agcaatgagg 121 tctaggaatg gtgttcttca tttattccttttcgttcttg catggcttct gttggcggct 181 ctccatgcta actcaagttc ggatgagagatcaacatata tagttcatat ggacaagacc 241 catatgccca aaaccttctc tagcccccaccattggtact cttcggtcgt tcgatccctc 301 aagtctacaa agccaaccaa attaaatcgccgtcgatcct caccacttct tgtatactct 361 tacgacaatg ctgctcatgg tttcagtgcagttttatctc aacaggaact tgaaactcta 421 aaaaagtctc caggtttcgt ctcagtttatgccgataaga cagcgacact tgacaccacc 481 catacacctg aatttctctc cctgaatactgccaacgggt tgtggcctgc ttcaaagtat 541 ggtgaagata taattgttgg tgttattgacagcggtgtct ggccggagag tgaaagttat 601 aatgatgatg gtatgggcgc tattccaagcagatggaagg gagaatgtga agctggacaa 661 gagttcaatt cctccatgtg caactcaaagcttattggag ctagatattt cgataagggt 721 atcattgcgg caaatcctgg gattaacattagcatgaaat ctgccagaga tactatgggg 781 catgggactc acacatcctc cacagttgctgggaattatg tggatggcgt ttcattcttt 841 ggctatgcta aaggtacagc aaaaggagtggcaccacggg cgagagtggc tatgtacaag 901 gtcatttttg acgaagggcg ctatgcatctgatgttcttg ccggtatgga cgcggctatt 961 gctgatggtg ttgatgtaat ttcaatatcaatgggatttg atgagacccc gttgtatgaa 1021 gatcctatag caattgcctc attcgctgctacagagaagg gcgtagtggt ctcatcttca 1081 gcaggaaatg cagggccagc gctagggagcttgcacaatg gaatcccatg gacgttaact 1141 gttgcagctg gaaccattga ccgttcatttgcaggcacta taactcttgg gagtggggaa 1201 accatcattg gatggacaat gttcccagccagtgcttatg tagcagactt gccactgctt 1261 tataacaaga cttactctgc atgcaactcaactcgattat tatctcaact ccgaactgac 1321 gccatcatcg tatgcgaaga agctgaagattcggtatctg agcaaatatc tgttgtcagt 1381 gcatcgaaca ttcggggagc catatttgtttcagattatg atgctgaatt atttgaactt 1441 ggtggtgtga ctattcctgg tgtcgtgattagcaccaagg atgcaccggc tgtgatcagc 1501 tacgccagca atgatgtgaa acctaaggcaagcatcaagt tccaacaaac tgttctgggc 1561 acaaagcctg caccagccgt ggctttctatacttctagag gtccgtcacc gagctatcca 1621 ggcatcttaa agccagatat aatggcccctgggtcactag tttttgctgc ttggattcca 1681 aatactgcta cagcccaaat tggtttgaataccctcttga caagtgaata caatatggtt 1741 tctggaacat caatggcctg ccctcatgctgctggtgtag ctgctctcct taagggcgca 1801 caccctgaat ggagtgcagc tgctattaggtctgcaatga tgactacagc aaatcccttg 1861 gataacacac taaatccaat ccgggacaatggtctaatca atttcacatc tgcttcacct 1921 ttagctatgg gagccggcca agttgatcctaatcgggcac ttgatcctgg tttgatttat 1981 gaaaccaccc cacaagatta tgtgagcctcctctgcactc tgaacttcac ccaaaaccaa 2041 atcctgtcca ttacaagatc aaaccgttacagctgctcca cccctaatcc tgatcttaac 2101 tatccttctt ttattacttt acactacaacacaaatgcaa catttgttca gacttttcac 2161 aggactgtga ctaacgttgg aggaagcgctacaacttaca aggccaagat cactgctcct 2221 ctaggttctg tagttagtgt ctcaccagacacattggcct tcagaaagca gtatgagcag 2281 cagagctacg agctcactat tgagtacaagcctgatggtg aagaaactgt ttcatttggg 2341 gaacttgttt ggattgaaga aaatgggaatcacactgtga ggagccctat tacagtgtca 2401 ccttccatga gtaactttgt gtttatgggtacacaataat tgataaaaat ttgttctgat 2461 cacaactgtg ggaataatcg acgtttatgaacccagaata agttgtttgg tcgtcttcaa 2521 cattatcata aaggacttga atcatgtgtgttgattttct gcaaaaaaaa aaaaaaaaaa 2581 aaagtactct gcgttgatac cactgcttgccctatagtga gtcgtattag

In the sequence SEQ. ID. NO. 8, an open reading frame is observed in thepresence of an initiation codon (ATG) encoding for an initiatormethionine in position 115 and a stop codon (UAA) in position 2437. Thepolynucleotide containing the sequence encoding for heterocarpinecorresponds to the sequence SEQ. ID. NO. 9, reproduced hereafter: SEQ.ID. NO. 9: 1 atgaggtcta ggaatggtgt tcttcattta ttccttttcg ttcttgcatggcttctgttg 61 gcggctctcc atgctaactc aagttcggat gagagatcaa catatatagttcatatggac 121 aagacccata tgcccaaaac cttctctagc ccccaccatt ggtactcttcggtcgttcga 181 tccctcaagt ctacaaagcc aaccaaatta aatcgccgtc gatcctcaccacttcttgta 241 tactcttacg acaatgctgc tcatggtttc agtgcagttt tatctcaacaggaacttgaa 301 actctaaaaa agtctccagg tttcgtctca gtttatgccg ataagacagcgacacttgac 361 accacccata cacctgaatt tctctccctg aatactgcca acgggttgtggcctgcttca 421 aagtatggtg aagatataat tgttggtgtt attgacagcg gtgtctggccggagagtgaa 481 agttataatg atgatggtat gggcgctatt ccaagcagat ggaagggagaatgtgaagct 541 ggacaagagt tcaattcctc catgtgcaac tcaaagctta ttggagctagatatttcgat 601 aagggtatca ttgcggcaaa tcctgggatt aacattagca tgaaatctgccagagatact 661 atggggcatg ggactcacac atcctccaca gttgctggga attatgtggatggcgtttca 721 ttctttggct atgctaaagg tacagcaaaa ggagtggcac cacgggcgagagtggctatg 781 tacaaggtca tttttgacga agggcgctat gcatctgatg ttcttgccggtatggacgcg 841 gctattgctg atggtgttga tgtaatttca atatcaatgg gatttgatgagaccccgttg 901 tatgaagatc ctatagcaat tgcctcattc gctgctacag agaagggcgtagtggtctca 961 tcttcagcag gaaatgcagg gccagcgcta gggagcttgc acaatggaatcccatggacg 1021 ttaactgttg cagctggaac cattgaccgt tcatttgcag gcactataactcttgggagt 1081 ggggaaacca tcattggatg gacaatgttc ccagccagtg cttatgtagcagacttgcca 1141 ctgctttata acaagactta ctctgcatgc aactcaactc gattattatctcaactccga 1201 actgacgcca tcatcgtatg cgaagaagct gaagattcgg tatctgagcaaatatctgtt 1261 gtcagtgcat cgaacattcg gggagccata tttgtttcag attatgatgctgaattattt 1321 gaacttggtg gtgtgactat tcctggtgtc gtgattagca ccaaggatgcaccggctgtg 1381 atcagctacg ccagcaatga tgtgaaacct aaggcaagca tcaagttccaacaaactgtt 1441 ctgggcacaa agcctgcacc agccgtggct ttctatactt ctagaggtccgtcaccgagc 1501 tatccaggca tcttaaagcc agatataatg gcccctgggt cactagtttttgctgcttgg 1561 attccaaata ctgctacagc ccaaattggt ttgaataccc tcttgacaagtgaatacaat 1621 atggtttctg gaacatcaat ggcctgccct catgctgctg gtgtagctgctctccttaag 1681 ggcgcacacc ctgaatggag tgcagctgct attaggtctg caatgatgactacagcaaat 1741 cccttggata acacactaaa tccaatccgg gacaatggtc taatcaatttcacatctgct 1801 tcacctttag ctatgggagc cggccaagtt gatcctaatc gggcacttgatcctggtttg 1861 atttatgaaa ccaccccaca agattatgtg agcctcctct gcactctgaacttcacccaa 1921 aaccaaatcc tgtccattac aagatcaaac cgttacagct gctccacccctaatcctgat 1981 cttaactatc cttcttttat tactttacac tacaacacaa atgcaacatttgttcagact 2041 tttcacagga ctgtgactaa cgttggagga agcgctacaa cttacaaggccaagatcact 2101 gctcctctag gttctgtagt tagtgtctca coagacacat tggccttcagaaagcagtat 2161 gagcagcaga gctacgagct cactattgag tacaagcctg atggtgaagaaactgtttca 2221 tttggggaac ttgtttggat tgaagaaaat gggaatcaca ctgtgaggagccctattaca 2281 gtgtcacctt ccatgagtaa ctttgtgttt atgggtacac aataa

A protein of sequence SEQ. ID. NO. 10, composed of 774 amino acids andreproduced hereafter corresponds to the polynucleotide thus translated:SEQ. ID. NO. 10: 1 M R S R N G V L H L F L F V L A W L L L A A L H A N SS S D 31 E R S T Y I V H M D K T H M P K T F S S P H H W Y S S V V R 61S L K S T K P T K L N R R R S S P L L V Y S Y D N A A H G F 91 S A V L SQ Q E L E T L K K S P G F V S V Y A D K T A T L D 121 T T H T P E F L SL N T A N G L W P A S K Y G E D I I V G V 151 I D S G V W P E S E S Y ND D G M G A I P S R W K G E C E A 181 G Q E F N S S M C N S K L I G A RY F D K G I I A A N P G I 211 N I S M K S A R D T M G H G T H T S S T VA G N Y V D G V S 241 F F G Y A K G T A K G V A P R A R V A M Y K V I FD E G R Y 271 A S D V L A G M D A A I A D G V D V I S I S M G F D E T PL 301 Y E D P I A I A S F A A T E K G V V V S S S A G N A G P A L 331 GS L H N G I P W T L T V A A G T I D R S F A G T I T L G S 361 G E T I IG W T M F P A S A Y V A D L P L L Y N K T Y S A C 391 N S T R L L S Q LR T D A I I V C E E A E D S V S E Q I S V 421 V S A S N I R G A I F V SD Y D A E L F E L G G V T I P G V 451 V I S T K D A P A V I S Y A S N DV K P K A S I K F Q Q T V 481 L G T K P A P A V A F Y T S R G P S P S YP G I L K P D I M 511 A P G S L V F A A W I P N T A T A Q I G L N T L LT S E Y N 541 M V S G T S M A C P H A A G V A A L L K G A H P E W S A AA 571 I R S A M M T T A N P L D N T L N P I R D N G L I N F T S A 601 SP L A M G A G Q V D P N R A L D P G L I Y E T T P Q D Y V 631 S L L C TL N F T Q N Q I L S I T R S N R Y S C S T P N P D 661 L N Y P S F I T LH Y N T N A T F V Q T F H R T V T N V G G 691 S A T T Y K A K I T A P LG S V V S V S P D T L A F R K Q Y 721 E Q Q S Y E L T I E Y K P D G E ET V S F G E L V W I E E N 751 G N H T V R S P I T V S P S M S N F V F MG T Q

Example 2 Preparation of the Complete cDNA Encoding for Heterocarpinefor Producing Recombinant Heterocarpine

2.1 ) Preparation of the RNAs from Pilocarous Heterophyllus CellCulture:

The cells in culture are preserved at −80° C. before the stages of totalRNA extraction. The total RNA extraction is based on a techniquedescribed in the scientific literature (Chomczynski and Sacchi, Anal.Biochem. (1987), 162, 156) using Trizol reagent (Gibco/BRL). The qualityof the RNAs thus extracted is analyzed on 1% agarose gel in the presenceof ethidium bromide.

2.2) Reverse Transcription from the RNAs:

The total RNAs are reverse transcribed with Oligo(dT) primers usingSuperscript® reverse transcriptase as suggested in the manufacturer's(Gibco/BRL) manual.

2.3) Polymerase Chain Reaction (PCR) on the Products Obtained FromReverse Transcription:

The amplification of the cDNA of heterocarpine was carried out bypolymerase chain reaction (PCR) on the products of reverse transcriptionusing the primers Fwd2 and Rev2 of respective sequences SEQ. ID. NO. 11and SEQ. ID. NO. 12.

The sequences SEQ. ID. NO. 11 and SEQ. ID. NO. 12 are the following:SEQ. ID. NO. 11: 5′-GGG GGA TCC GAG GTC TAG GAA TGG TGT TCT TCA-3′ SEQ.ID. NO. 12: 5′-GGG CTC GAG TTG TGT ACC CAT AAA CAC AAA GTT ACT CAT GG-3′

The primer Fwd2 corresponds to the nucleotides 118 to 140 of SEQ. ID.NO. 8 and includes a BamH1 site (underlined) and three additionalnucleotides in the 5′ part in order to facilitate the cloning. Theprimer Rev2 corresponds to the sequence complementary to the regioncontaining the nucleotides 2405 to 2436 of SEQ. ID. NO. 8 and includesan Xhol site (underlined) and three additional nucleotides in the 5′part in order to facilitate the cloning.

The reaction conditions include 50 ng of the cDNA products of thereverse transcription reaction described above, 0.2 μM of Fwd2 (SEQ. ID.NO. 11) and of Rev2 (SEQ. ID. NO. 12), 200 μM dNTPs, 40 mM Tricine-KOH(pH 8.7), 15 mM KOAc, 3.5 mM Mg(Oac)₂, 3.75 μg/ml BSA, 0.005% Tween-20,0.005% Nonidet-P40, and 0.5 U Taq DNA polymerase in a final volume of 50μl. The PCR reactions are carried out in a Perkin-Elmer 9700thermocycler using the following thermal cycle parameters: 5 cyclescomprising a denaturation at 94° C. for 5 seconds, a hybridization ofthe primers at 72° C., 5 cycles comprising a denaturation at 94° C. for5 seconds, a hybridization of the primers at 70° C. for 10 seconds, anda polymerase extension at 72° C. for 3 minutes and finally 25 cyclescomprising a denaturation at 94° C. for 5 seconds, a hybridization ofthe primers at 68° C. for 10 seconds, and a polymerase extension at 72°C. for 3 minutes.

The products obtained by PCR are separated on 1% agarose gel andvisualized using ethidium bromide staining. An approximately 2.3 kb bandis obtained.

The nucleic acid sequence of the PCR product is verified using anautomatic sequencer and corresponds to the sequence SEQ. ID. NO. 9having artificially undergone deletion of the initiation codon ATG inorder to allow the expression of recombinant heterocarpine from thepQE-TriSystem (Qiagen) phase vector with the BamH1 site as well asdeletion of the stop codon in order to preserve the phase translation toprotein and thus allow the synthesis of an 8× His sequence in theC-terminal region of heterocarpine. This sequence corresponds to thesequence SEQ. ID. NO. 13 reproduced hereafter: SEQ. ID. NO. 13: 1gggggatccg aggtctagga atggtgttct tcatttattc cttttcgttc ttgcatggct 61tctgttggcg gctctccatg ctaactcaag ttcggatgag agatcaacat atatagttca 121tatggacaag acccatatgc ccaaaacctt ctctagcccc caccattggt actcttcggt 181cgttcgatcc ctcaagtcta caaagccaac caaattaaat cgccgtcgat cctcaccact 241tcttgtatac tcttacgaca atgctgctca tggtttcagt gcagttttat ctcaacagga 301acttgaaact ctaaaaaagt ctccaggttt cgtctcagtt tatgccgata agacagcgac 361acttgacacc acccatacac ctgaatttct ctccctgaat actgccaacg ggttgtggcc 421tgcttcaaag tatggtgaag atataattgt tggtgttatt gacagcggtg tctggccgga 481gagtgaaagt tataatgatg atggtatggg cgctattcca agcagatgga agggagaatg 541tgaagctgga caagagttca attcctccat gtgcaactca aagcttattg gagctagata 601tttcgataag ggtatcattg cggcaaatcc tgggattaac attagcatga aatctgccag 661agatactatg gggcatggga ctcacacatc ctccacagtt gctgggaatt atgtggatgg 721cgtttcattc tttggctatg ctaaaggtac agcaaaagga gtggcaccac gggcgagagt 781ggctatgtac aaggtcattt ttgacgaagg gcgctatgca tctgatgttc ttgccggtat 841ggacgcggct attgctgatg gtgttgatgt aatttcaata tcaatgggat ttgatgagac 901cccgttgtat gaagatccta tagcaattgc ctcattcgct gctacagaga agggcqtagt 961ggtctcatct tcagcaggaa atgcagggcc agcgctaggg agcttgcaca atggaatccc 1021atggacgtta actgttgcag ctggaaccat tgaccgttca tttgcaggca ctataactct 1081tgggagtggg gaaaccatca ttggatggac aatgttccca gccagtgctt atgtagcaga 1141cttgccactg ctttataaca agacttactc tgcatgcaac tcaactcgat tattatctca 1201actccgaact gacgccatca tcgtatgcga agaagctgaa gattcggtat ctgagcaaat 1261atctgttgtc agtgcatcga acattcgggg agccatattt gtttcagatt atgatgctga 1321attatttgaa cttggtggtg tgactattcc tggtgtcgtg attagcacca aggatgcacc 1381ggctgtgatc agctacgcca gcaatgatgt gaaacctaag gcaagcatca agttccaaca 1441aactgttctg ggcacaaagc ctgcaccagc cgtggctttc tatacttcta gaggtccgtc 1501accgagctat ccaggcatct taaagccaga tataatggcc cctgggtcac tagtttttgc 1561tgcttggatt ccaaatactg ctacagccca aattggtttg aataccctct tgacaagtga 1621atacaatatg gtttctggaa catcaatggc ctgccctcat gctgctggtg tagctgctct 1681ccttaagggc gcacaccctg aatggagtgc agctgctatt aggtctgcaa tgatgactac 1741agcaaatccc ttggataaca cactaaatcc aatccgggac aatggtctaa tcaatttcac 1801atctgcttca cctttagcta tgggagccgg ccaagttgat cctaatcggg cacttgatcc 1861tggtttgatt tatgaaacca ccccacaaga ttatgtgagc ctcctctgca ctctgaactt 1921cacccaaaac caaatcctgt ccattacaag atcaaaccgt tacagctgct ccacccctaa 1981tcctgatctt aactatcctt cttttattac tttacactac aacacaaatg caacatttgt 2041tcagactttt cacaggactg tgactaacgt tggaggaagc gctacaactt acaaggccaa 2101gatcactgct cctctaggtt ctgtagttag tgtctcacca gacacattgg ccttcagaaa 2161gcagtatgag cagcagagct acgagctcac tattgagtac aagcctgatg gtgaagaaac 2221tgtttcattt ggggaacttg tttggattga agaaaatggg aatcacactg tgaggagccc 2281tattacagtg tcaccttcca tgagtaactt tgtgtttatg ggtacacaac tcgagccc

This sequence encodes for a protein of sequence SEQ. ID. NO. 14reproduced hereafter: 1 M A I S R E L V D P R S R N G V L H L F L F V LA W L L L A 31 A L H A N S S S D E R S T Y I V H M D K T H M P K T F S SP 61 H H W Y S S V V R S L K S T K P T K L N R R R S S P L L V Y 91 S YD N A A H G F S A V L S Q Q E L E T L K K S P G F V S V 121 Y A D K T AT L D T T H T P E F L S L N T A N G L W P A S K 151 Y G E D I I V G V ID S G V W P E S E S Y N D D G M G A I P 181 S R W K G E C E A G Q E F NS S M C N S K L I G A R Y F D K 211 G I I A A N P G I N I S M K S A R DT M G H G T H T S S T V 241 A G N Y V D G V S F F G Y A K G T A K G V AP R A R V A M Y 271 K V I F D E G R Y A S D V L A G M D A A I A D G V DV I S I 301 S M G F D E T P L Y E D P I A I A S F A A T E K G V V V S S331 S A G N A G P A L G S L H N G I P W T L T V A A G T I D R S 361 F AG T I T L G S G E T I I G W T M F P A S A Y V A D L P L 391 L Y N K T YS A C N S T R L L S Q L R T D A I I V C E E A E 421 D S V S E Q I S V VS A S N I R G A I F V S D Y D A E L F E 451 L G G V T I P G V V I S T KD A P A V I S Y A S N D V K P K 481 A S I K F Q Q T V L G T K P A P A VA F Y T S R G P S P S Y 511 P G I L K P D I M A P G S L V F A A W I P NT A T A Q I G L 541 N T L L T S E Y N M V S G T S M A C P H A A G V A AL L K G 571 A H P E W S A A A I R S A M M T T A N P L D N T L N P I R D601 N G L I N F T S A S P L A M G A G Q V D P N R A L D P G L I 631 Y ET T P Q D Y V S L L C T L N F T Q N Q I L S I T R S N R 661 Y S C S T PN P D L N Y P S F I T L H Y N T N A T F V Q T F 691 H R T V T N V G G SA T T Y K A K I T A P L G S V V S V S P 721 D T L A F R K Q Y E Q Q S YE L T I E Y K P D G E E T V S F 751 G E L V W I E E N G N H T V R S P IT V S P S M S N F V F M 781 G T Q L E H H H H H H H H

Example 3 Production of Recombinant Heterocarpine By Bacteria

The part of the cDNA encoding for heterocarpine is inserted at theBamH1/Xho1 sites of the pQE-TriSystem (Qiagen) expression vector and isexpressed using E. coli M15 bacteria as host bacteria. 20 ml of LBmedium containing 100 μg/ml of ampicillin and 25 μg/ml of kanamycin areinoculated and the bacteria brought to 37° C. under stirring for 12hours. From this culture, 1 litre of LB medium containing 100 μg/ml ofampicillin and 25 μg/ml of kanamycin is inoculated and the bacteria arestirred at 37° C. until an optical density of 0.6 at 600 nm is obtained.

The expression of the recombinant heterocarpine is produced by theaddition of IPTG at a final concentration of 1 mM over 4 to 5 hours. Thebacteria are then recovered by centrifugation at 4000×g for 20 minutesthen frozen in liquid nitrogen. The pellet is then thawed in ice for 15minutes and suspended in a pH 8.0 lysis buffer composed of 50 mMNaH₂PO₄, 300 mM NaCl and 10 mM imidazole in the presence of 1 mg/ml oflysozyme for 30 minutes in ice. The lysis is finally completed by asonication stage and the debris eliminated by centrifugation. The lysate(4 ml) thus clarified is mixed with 1 ml of a nickel matrix suspensionand stirred at 4° C. for 60 minutes. All of the reaction medium isplaced in a column, washed in the presence of 50 mM NaH₂PO₄, 300 mM NaCland 20 mM imidazole. The heterocarpine is finally eluted with 4×0.5 mlof buffer constituted by 50 mM NaH₂PO₄, 300 mM NaCl and 250 mMimidazole.

Example 4 Production of Recombinant Heterocarpine By Cells From InsectsInfected By the Baculovirus

The part of the cDNA encoding for heterocarpine is inserted at theBamH1/Xho1 sites of the pQE-TriSystem (Qiagen) expression vector. ThepQE-TriSystem vector contains the viral sequences of Autographacalifornia nuclear polyhedrosis virus (AcNPV) allowing homologousrecombination. The recombinant baculovirus containing the heterocarpinesequence is prepared by co-transfection of the pQE-TriSystem vector withthe genomic linearized DNA of the baculovirus in sf9 or sf21 insectcells established from ovarian tissues of the Spodoptera frugiperdalarva. The transfected cells are washed with phosphate buffer andcollected by centrifugation at 1000×g for 5 minutes. The pellet issuspended in a pH 8.0 lysis buffer composed of 50 mM NaH₂PO₄, 300 mMNaCl and 10 mM imidazole. The lysis is finally completed by a sonicationstage and the debris eliminated by centrifugation. The lysate (4 ml)thus clarified is mixed with 200 μl of a nickel matrix suspension andstirred at 4° C. for 60 minutes. All of the reaction medium is placed ina column, washed in the presence of 50 mM NaH₂PO₄, 300 mM NaCl and 20 mMimidazole. The heterocarpine is finally eluted with 4×0.5 ml of bufferconstituted by 50 mM NaH₂PO₄, 300 mM NaCl and 250 mM imidazole.

Example 5 Production of Recombinant Heterocarpine By Mammal Cells

The part of the cDNA encoding for heterocarpine is inserted at theBamH1/Xho1 sites of the pQE-TriSystem (Qiagen) expression vector. ThepQE-TriSystem vector contains the activating sequences of thecytomegalovirus (CMV) fused to chicken beta-actin promoter allowing avery significant heterologous expression. Human embryo kidney (HEK-293)cells are cultured in DMEM medium (Dulbecco's Modified Eagle's Medium)containing 100 U/ml of penicillin and 100 μg/ml of streptomycinsulphate, complemented with 10% foetal calf serum. The cells aresub-cultured 24 hours before the transfection protocol allowing normalmetabolism of the cells and better transfection efficiency. Thetransfection of 1 μg of pQE-TriSystem containing the cDNA encoding forheterocarpine was carried out using Effectene® reagent according to themanufacturer's (Qiagen) recommendations.

The transfected cells are washed with phosphate buffer and collected bycentrifugation at 1000×g for 5 minutes. The pellet is suspended in a pH8.0 lysis buffer composed of 50 mM NaH₂PO₄, 300 mM NaCl, 10 mM imidazolein the presence of 0.05% Tween® 20. The lysis is finally completed by asonication stage and the debris eliminated by centrifugation. The lysate(4 ml) thus clarified is mixed with 200 μl of a nickel matrix suspensionand stirred at 4° C. for 60 minutes. All of the reaction medium isplaced in a column, washed in the presence of 50 mM NaH₂PO₄, 300 mM NaCland 20 mM imidazole. The heterocarpine is finally eluted with 4×0.5 mlof buffer constituted by 50 mM NaH₂PO₄, 300 mM NaCl and 250 mMimidazole.

Example 6 Measurement of the Binding to the Human GHRH Receptor

Stable Transfections of the Human GHRH Receptor (hGHRH-R):

HEK-293 human embryo kidney cells (a cell line developed by Dr. StuartSealfon, Mount Sinai Medical School, New York, N.Y.) expressing thehuman GHRH receptor in stable manner were obtained from Dr. Kelly Mayo(Northwestern University, Chicago, Ill.).

Cell Culture and Membrane Preparation:

The HEK-293 cells transfected in a stable manner with the human GHRHreceptor described above are cultured in DMEM (Dulbecco's ModifiedEagle's Medium, high glucose content; supplied by Life technologies)supplemented with 0.4 mg/ml of G418 (Life technologies) in the presenceof 10% foetal calf serum and 4 mM of L-glutamine (Life Technologies).The cells are homogenized in buffer A containing 50 mM HEPES (pH 7.4), 5mM of magnesium chloride (MgCl₂), 2 mM ofethyleneglycol-bis(2-amino-ethyl)-N,N,N′,N′-tetraacetic acid (EGTA) and50 μg/ml of bacitracin then subjected to sonication in the same bufferA. The cells thus homogenized are centrifuged at 4° C. at 39,000 g for10 minutes, suspended in the buffer A and re-centrifuged at 4° C. at40,000 g for 10 minutes. The total membrane proteins are quantified byBradford's technique. The pelleted membranes are thus stored at −80° C.for later use.

Competitive Binding Test on hGHRH-R:

The membranes of the HEK-293 cells transfected in a stable manner withthe human GHRH receptor are diluted to a concentration of 100 μg/ml inreaction buffer containing 50 mM HEPES (pH 7.4), 5 mM of MgCl₂, 2 mM ofEGTA, 50 μg/ml of bacitracin and 0.5% bovine serum albumin (BSA). Themembranes are incubated with 0.05 nM of [¹²⁵I]GHRH(1-44 amide)(Amersham) in a final volume of 200 μl in the presence of increasingconcentrations of heterocarpine for 2 hours at 23° C. The reaction isstopped by rapid filtration on GF/C 96-well filters 0.1% pre-loaded withpolyethylenimine. The filters are then washed three times at 4° C. withwashing buffer containing 50 mM Tris (pH 7.4) using a Packard 96-wellfiltration station. The filters thus dried are submerged in 20 μl ofscintillation cocktail (Microscint O, Packard) and are subjected tocounting by Topcount (Packard). The non-specific activity is determinedin the presence of 100 nM of hGHRH. A dose-response curve is generatedfor hGHRH (0.001 nM-100 nM) and makes it possible to determine theinhibitory concentration IC₅₀ of protein/polypeptide at which 50% humanGHRH does not bind to human GHRH receptor.

1. Isolated polynucleotide comprising the sequence SEQ.ID.NO. 8 or oneof its fragments.
 2. An isolated polynucleotide according to claim 1,wherein it is a polynucleotide of sequence SEQ.ID.NO.8.
 3. An isolatedpolynucleotide according to claim 1, wherein it is a polynucleotide ofsequence SEQ.ID.NO.9.
 4. A polynucleotide selected from the groupconsisting of sequence SEQ.ID.NO.4, SEQ.ID.NO.5, SEQ.ID.NO.11 andSEQ.ID.NO.12.
 5. A polynucleotide of sequence SEQ.ID.NO.13.
 6. Anisolated polypeptide comprising the sequence SEQ.ID.NO.14 or one of itsfragments.
 7. An isolated polypeptide according to claim 6, wherein itis a polypeptide of sequence SEQ. ID.14.
 8. An expression vectorcontaining a polynucleotide of sequence SEQ.ID.NO.13.
 9. A host celltransformed or transfected by an expression vector according to claim 8.10. A process for preparing an isolated polypeptide comprising theprotein encoded by the polynucleotide sequence SEQ.ID.NO.9 orSEQ.ID.NO.13 or one of the fragements of the latter or by a sequencecomplementary to the polynucleotide sequence SEQ.ID.NO.9 or one of thefragments of the latter, said isolated polypeptide having at least oneimmunological an./or biological activity characteristic of a proteinbinding human GHRH and being associated with the modulation of cellproliferation, said preparation process comprising the following steps:(a) culture, under suitable conditions to obtain the expression of saidpolypeptide of a host cell transformed or transfected with an expressionvector comprising an isolated polynucleotide comprising thepolynucleotide sequence SEQ.ID.NO.9 or SEQ.ID.NO.13, the sequencecomplementary to the polnucleotide sequence SEQ.ID.NO.9 or SEQ.ID.NO.13or also one of the fragments of the latter, said isolated polypeptidehaving at least one immunological and/or biological activitycharteristic of a protein human GHRN and being associated with themodulation of cell proliferation, and (b) isolation of the polypeptidefrom the host cell cultures.
 11. An antibody or antigen-binding fragmentof the latter, which specifically binds the protein sequenceSEQ.ID.NO.14 but not the protein of sequence SEQ.ID.NO.10.
 12. Asmedicament, a polynucleotide according to claim
 1. 13. As medication, apolypeptide according to claim
 6. 14. Pharmaceutical compositioncomprising, as active ingredient, a polynucleotide according to claim 1.15. Pharmaceutical composition comprising, as active ingredient, apolypeptide according to claim 6 or
 7. 16. Use of a polynucleotideaccording to claim 1 for preparing a medicament intended to treat aproliferative disease.
 17. Use of a polypeptide according to claim 6 forpreparing a medicament intended to treat a proliferative disease.
 18. Amethod for the identification of compounds capable of binding human GHRHand modulating cell proliferation, comprising: (a) bring each candidatecompound into contact with an isolated polypeptide comprising: either afragment of the protein encoded by the polynucleotide sequenceSEQ.ID.NO.9 or by a sequence complementary to the polynucleotidesequence SEQ.ID.NO.9, or a fragment of the protein encoded by thepolynucleotide sequence SEQ.ID.NO.13 or by a sequence complementary tothe polynucleotide sequence SEQ.ID.NO.13, under condition and for a timesufficient to allow the candidate agent to bind to the polypeptide, saidisolated polypeptide having at least one immunological and/or biologicalactivity characteristic of a protein binding human GHRH and beingassociated with the modulation of cell proliferation, and (b) detectionof the binding of each candidate compound to said polypeptide andidentification, from the candidate compounds, of the compounds capableof binding human GHRH and modulating cell proliferation.
 19. Apharmaceutical composition for treating a proliferative diseasecomprising an amount of a polynucleotide of claim 1 sufficient to treatsaid disease and an inert carrier.
 20. A pharmaceutical composition fortreating a proliferative disease comprising an amount of a polypeptideof claim 6 sufficient to treat said disease and an inert carrier.
 21. Amethod of treating a proliferative disease in a warm-blooded animalcomprising administrating to a warm-blooded animal an amount ofpolynucleotide of claim 1 sufficient to treat said disease.
 22. A methodof treating a proliferative disease in a warm-blooded animal comprisingadministrating to a warm-blooded animal an amount of polypeptide ofclaim 6 sufficient to treat said disease.